Enhancement of photocatalytic and biological activities of chitosan/activated carbon incorporated with TiO2 nanoparticles

Novel and sustainable chitosan (CS)/activated charcoal (AC) composites were prepared by cross-linking with epichlorohydrin (ECH) to form a porous structure. Different titanium dioxide nanoparticle (TiO2 NPs) concentrations (0, 0.2, 0.4, and 0.8% w/w) were added to enhance the photocatalytic, antibacterial, larvicidal, and pupicidal activities’ efficiency toward Rose Bengal (RB) dye and the Culex pipiens. The composites were characterized by FT-IR, XRD, XPS, BET and SEM. The SEM images revealed the porous structure of CS/AC and TiO2 nanoparticles were uniformly distributed in the CS/AC matrix. The degradation of RB dye was used to test the photocatalytic behavior of the composites. Supporting TiO2 on a CS/AC matrix resulted in a significant increase in photocatalytic performance. The antibacterial activities supported by CS/AC/TiO2 NPs were evaluated by bacterial growth inhibition against B. subtilis, S. aureus, E. coli, and P. aeruginosa. The results showed that CS/AC/TiO2 NPs composite has an inhibitory effect and therefore considered antibacterial agents. CS/AC/0.4%TiO2 NPs showed maximum efficacy against larvicidal activity and pupicidal of mosquito vector which recorded 99.00 ± 1.14, 95.00 ± 1.43, and 92.20 ± 2.64 for the first, second, and third larval instars and 66.00 ± 2.39 for pupal mortality, while the repellent activity reported high protection at 82.95 ± 2.99 with 3.24 mg/cm2 dose compared to control DEET.


Introduction
Chitosan (CS) is a linear heteropolysaccharide consisting of n-acetylglucosamine and 1,4-β-glucosamine, which is derived from the deacetylation of chitin (Aragunde et al. 2018). It is insoluble in water as a result of the bonds between the polymer chains with hydrophilic nature, nontoxicity, and biodegradability (Cheung et al. 2015). Moreover, it is structure contains many free and active groups of (-NH 2 ) and (-OH) that can form coordination bonds with the empty orbital of metal elements (Xiong et al. 2013;Zhao et al. 2013;Luo et al. 2015). Therefore, CS biopolymer has been widely used to remove dyes and metal ions and become increasingly important in environmental biotechnology. CS as adsorbents has the advantages of being low cost, renewable, abundant, ecofriendly, multifunctional, and non-toxic and causing no secondary pollution. However, the use of CS as an adsorbent in wastewater treatment technologies is still limited due to its high solubility in an acidic medium, filtration ability, poor mechanical properties, and swelling of the aqueous medium. However, due to its high solubility in an acidic medium, filtration efficiency, poor mechanical properties, and swelling of the aqueous medium, the use of CS as an adsorbent in wastewater treatment technologies is still limited. A very effective way to overcome these limitations and enhance the physiochemical properties of the CS biopolymer is chemical modification by cross-linking reaction using epichlorohydrin (ECH) with activated charcoal (AC) in the presence of metal oxides such as TiO 2 . AC Responsible Editor: Sami Rtimi * Ragab E. Abouzeid r_abouzeid2002@yahoo.com 1 is also commonly used in water treatment, although it has some technological drawbacks, including a high cost and difficulty in recovering it (Jung et al. 2016a, b;Hassan et al. 2017). AC particles have been encapsulated in a polymeric support, predominantly CS, to overcome these drawbacks. The use of this biological macromolecule produces composites with lower costs, better mechanical properties, and a higher number of functional groups (Afzal et al. 2018). TiO 2 is an inert, nontoxic, chemically stable, biocompatible, and cheap material having been approved as safe for use in food and materials in contact with food by the Food and Drug Administration (FDA) TiO 2 is a nontoxic, chemically stable, biocompatible, and inexpensive substance that has been licenced by the Food and Drug Administration for the safe use in food and products that come into contact with food (FDA) (Zhou et al. 2009;Othman et al. 2014;El-Wakil et al. 2015;El-Gendy et al. 2017;Zhang et al. 2017). The photocatalytic activity of TiO 2 nanoparticles is higher than that of bulk TiO 2 particles due to its high surface area to volume ratio. When exposed to ultraviolet (UV) light, TiO 2 nanoparticles have a higher band gap energy and generates electron-hole pairs on their surface, which improves their efficiency Due to their high surface area to volume ratio, TiO 2 nanoparticles have a higher photocatalytic activity than bulk TiO 2 particles. TiO 2 nanoparticles have a higher band gap energy and produce electron-hole pairs on their surface when exposed to UV light, which improves their performance. (Maneerat et al. 2003;Onda et al. 2005;Lian et al. 2016). However, the tendency of nanosized TiO 2 to agglomerate reduces its performance (Qian et al. 2011;Lin et al. 2015). This random agglomeration of TiO 2 nanoparticles reduces the surface area of the particles and lowering their photocatalytic activity (Li et al. 2010;Lin et al. 2015). Photodegradation of organic pollutants, gas sensor, lithium battery processing, white pigments, wound healing, and drug delivery systems have been described as applications for TiO 2 NPs (Ali et al. 2015;Zhang et al. 2017;Abouzeid et al. 2019;Elfeky et al. 2020). The addition of TiO 2 NPs to food packaging films has been shown to enhance the films' physical, thermal, and mechanical properties (Xing et al. 2012 (Zhang et al. 2021). Mosquitoes are responsible for the transportation of many pathogenic agents to humans worldwide (Benelli et al. 2018). C. pipiens L. (Diptera: Cuicidae) was recorded as one of the most widely dispensed mosquitoes in the world. It is called house mosquito and is widely distributed in the urban areas and lives near people (Bernard et al. 2001;Oz et al. 2013). From this importance, the research today diverted to find alternative agents that possess bioactive chemicals which act as insecticides, repellents as well as growth inhibitors (Murugan et al. 1996). The aim of this study was to produce crosslinked CS/AC/ by ECH and then incorporated with different concentrations of TiO 2 NPs as a suitable candidate for the removal of anionic dyes, such as Rose Bengal (RB) dye, assess its photodegradation properties and to evaluate the biological activities of the composites.

Materials
Chitosan (80-95% deacetylated, average molecular weight (MW) 9.0 × 106 Da), titanium dioxide (TiO 2 ) nanoparticles activated charcoal and Epichlorohydrin were purchased from Sigma-Aldrich. All the other chemicals were of analytical reagent grades and used without further purification.

Preparation of the composite CS/AC/ TiO 2 NPs
Chitosan solution (1% w/v) was prepared by dissolving 1 g of CS powder in 100 ml of a 1% (w/w) aqueous solution of acetic acid and stirred on a magnetic stirrer for at least 3 h at room temperature (until the solution became homogenous). Then half gram of AC was added to the CS solution until reaching a more uniform dispersion by ultrasound for 20, min then 1 ml of ECH aqueous solution was added and stirred vigorously for cross-linked reaction for another 1 h. Different concentrations of TiO 2 NPs were added to the solution and stirring for 30 min. The final product was obtained by immersed in a coagulation bath (50 ml of sodium hydroxide solution (5 mmol L −1 )) for 2 h, then the samples were separated from the solution by centrifugation at 10,000 rpm for 10 min and washed with deionized water. The obtained composite was dried at 100 °C, then ground in a mortar to a homogenous fine powder and used for photodegradation and biological studies. The possible mechanism was shown in Scheme 1

Characterization
The morphology structure was carried out by scanning electron microscopy (SEM, Quanta-250 conducted with EDAX). Fourier transform infrared spectroscopy (FT-IR) was performed by the KBr method on a Mattson 5000 spectrometer (Unicam, UK). The X-ray diffraction (XRD) was identified using X-ray diffractometer (PANa-lytical, Netherlands) at 25 °C with Cu Kα a monochromatic radiation source (λ = 0.154 nm, 2θ = 5°: 80°, and scanning time 5 min). X-ray photoelectron spectroscopy (XPS) spectra were collected on an ESCALAB 250XI + instrument (Thermo Scientific, KALPHA, UK) with monochromatic X-ray Al Kα radiation (1486.6 eV) and the following operating parameters: spot size, 500 μm; absolute resolved energy interval calibrated with Ag3d5/2 line (0.45 eV) and C1 s line (0.82 eV); sample preparation pressure 10-8 mbar; full spectrum pass energy 50 eV, and narrow-spectrum pass energy 20 eV. Data were analyzed with Casa XPS processing and MultiPak software. The surface area and pore structure of the CS/AC and CS/ AC/TiO 2 were calculated by N 2 adsorption/desorption isotherms using a Quantachrome Instruments analyzer (St 2 on NOVA Touch 4LX device).

Photodegradation procedure
The photocatalytic activity of CS/AC and CS/AC/TiO 2 is assessed utilizing the photocatalytic degradation of RB as a reaction probe in a beaker with stirring. For photocatalytic experiments, 100 ml (7.9 ppm) solution RB (C20H2Cl4I-4Na2O5, M.Wt. 1017.65, LABA Chemie, CI # 45,440) (Scheme 2) was taken into the reactor with the required amount of the catalyst. Prior to irradiation, the solution was attractively blended in obscurity for 10 min to accomplish the adsorption equilibration of the framework. After irradiations utilizing UV-lamp (20 W), testing samples were removed at various time intervals, filtered, and afterward poured in a quartz cell. The RB concentration was determined by utilizing a Lambda UV/vis spectrophotometer (Perkin Elmer) at λ = 540 nm at different times. All photocatalytic reactions were performed at room temperature. The photodegradation efficiency was calculated by the following equation: where C 0 is the initial concentration of dye and C e is the final concentration of dye after illumination by UV-light.

Antibacterial activity
Antibacterial activity of the prepared composites (CS/ AC) at three different concentrations of TiO 2 NPs (0.2, 0.4, and 0.8) was investigated quantitatively by using the broth (turbidity) inhibition method depending on the optical density (OD), against Gram-negative Escherichia coli ATCC 8739 (E. coli) and Pseudomonas aeruginosa ATCC 9027 (P. aeruginosa) and Gram-positive Bacillus subtilis ATCC6633 (B. subtilis) Staphylococcus aureus ATCC25923 (S. aureus), as reference strains for antibacterial assay, obtained from the American Type Culture Collection (ATCC). Broth inhibition approach was conducted in a flask (50 ml) containing 10 ml of Luria-Bertani (LB) broth media which composed of (g/l): peptone 10, yeast extract 5, and NaCl 5. To prepare the Scheme 1 The possible interaction between CS-ECH/AC and TiO 2 Scheme 2 Molecular structure of RB salt: quinoid (q) form preinoclume, the strains were grown in LB broth at 30 °C and 200 rpm for 24 h. After cultivation time, each flask containing 10 ml of LB media was inoculated (100 μL) with the model bacteria used (OD at 660 nm reached 0.3) and assessed for antibacterial by adding 100 mg of the prepared composites, then incubated in a shaking incubator 200 rpm at 30 °C for 24 h. The control was performed by growing the model bacteria at the same conditions without adding the composites. After time consumed, the bacterial growth was measured by taking the OD at 600 nm and the results will be expressed as growth inhibition % by the formula (Adel et al. 2019): All the required materials used in this experiment are autoclaved. Triplicate experiments were carried out for each composite.

Larvicidal and pupicidal activity
The larvicidal and pupicidal activity of synthesized CS/ AC/TiO 2 (0.2, 0.4, and 0.8%) against the C. pipiens was Growth inhibition % = (OD control − OD sample)∕OD control × 100 evaluated by using the standard method bioassays (Tables 1, 2, and 3) (Williams et al. 1986). Each concentration was made separately at five different test concentrations (2, 4, 6, 8, and 10 ppm) of aqueous CS/AC/TiO 2 (0.2, 0.4, and 0.8%). To evaluate the larvicidal and pupicidal activity of synthesized CS/AC/TiO 2 (0.2, 0.4, and 0.8%), 25 larvae and pupae of Cx. Pipiens were separately and exposed to 100 ml of test concentrations. Also, the control (without CS/AC/ TiO 2 ) was run to test the normal mortality. After that time, the mortality, which was particular after different hours of treatment, at the time of the experiment. No food was given to the larvae during the experiment. The experiments were repeated five times to confirm the results. The performance data was subjected to probate analysis. Mortality was corrected with Abbott's formula.

Repellency activity
Standard cages (30 × 30 × 30 cm) were applied to test the repellent activity of the selected material. Different concentrations from the CS/AC/TiO 2 were directly applied onto 5 × 6 cm of abdomen surface of pigeon after feathers removal from the abdomen to correct the repellency with C. pipiens compared with commercial repellent DEET (N. N. diethyl-meta-roulamide) (Johnson Wax Egypt) as a positive control. After 10 min, the treated pigeons were placed in the cages containing 50 C. pipiens starved females for 3 h. Each test was repeated three times to get a mean value of repellent activity (Shehata 2019). After treatments, the number of unfed females was counted and calculated according: where: A = percent of unfed females in treatment B = percent of unfed females in control Figure 1a shows that the surface morphology of CS, CS/AC, and CS/AC/TiO 2 , the surface structure of CS smooth, and nonporous while the SEM image of CS/AC crosslinked with ECH was a rough and heterogonous surface with evident cavities and irregular pore size (Fig. 1b). In Fig. 1c, the TiO 2 NPs were distributed uniformly in the matrix as evidenced by images of the composites, with some agglomeration and porous structure which providing the surface area that can play an essential role in the photodegradation of RB dye.

FT-IR analysis
The functional groups of CS/AC and CS/AC/TiO 2 spectrum were determined using FT-IR analysis which revealed the following characteristic peaks in Fig. 2 The band at 450 cm −1 is due to the bending vibration of Ti-O-Ti bonds (Bineesh et al. 2010). These peaks confirmed that the AC was successfully grafted with the chains of CS-ECH with TiO 2 by the chemical interactions, such as hydrogen bonding formation between oxygen groups of AC and functional groups of the CS (Sharififard et al. 2018). The presence of amide, amine, and hydroxyl functional groups together with TiO 2 metal oxide confirms that it well-mixed and supports to effective dye removal through the RB dye photodegradation process.

XRD analysis
XRD patterns of CS/AC, CS/AC/TiO 2 are shown in Fig. 3. As we can see from the XRD of CS/AC, two peaks are appearing at about 25° and 42° corresponding to reflection

XPS analysis
In order to study the elementary composition on the surface of CS/AC/TiO 2 , X-ray photoelectron spectroscopy (XPS) was carried out. According to Fig.4A for the wide scan spectra of the three specimens, the peaks at 284.93, 398.5, and 399.06 eV correspond to C 1 s, N 1 s, and O 1 s, respectively (Fig. 4). The Ti 2p peak is found in the spectrum of CS/AC/TiO 2 (457.11 eV). Based on these results, N atoms had been incorporated into TiO 2 nanoparticles.  Figure 5 illustrates the N 2 adsorption/desorption isotherms which were used to determine the pore structure and specific surface area of CS/AC and CS/AC/TiO 2 . It was found that CS/AC and CS/AC/TiO 2 had surface areas of respectively 216.043 and 891 m 2 /g. There is a noticeable increase in the surface area of composites due to TiO 2 nanoparticles. The mean pore diameters of CS/AC and CS/AC/TiO 2 in Table 4 were 1.92 and 1.93 nm, respectively, indicating that they are mesoporous materials (Su et al. 2019). This high surface area and porous structure are essential for photodegradation to occur. Isotherms of N 2 adsorption on CS/AC and CS/AC/TiO 2 are classified as type IV in accordance with the IUPAC classification based on mesoporous adsorption isotherms (Fig. 5) (Yan et al. 2017).

Effect of AC/CS, TiO 2 , and AC/CS/TiO 2 components on RB degradation
Firstly, the three components will be examined individually and then together to determine the viability to enhance the catalytic performance.0.1 g photocatalysts were added into 100 RB solutions and 7.9 ppm dye concentration. As shown in Fig. 6, the photodegradation percentages are 73.1, 75.3, and 91.9% for samples CS/AC, TiO 2 , and CS/AC/TiO 2 NPs, respectively. The composite demonstrated higher photocatalytic activity than the CS/AC and TiO 2 under UV light irradiation. This occurrence is believed to be related to the higher degree of interphase contact that can be achieved at the TiO 2 surface with activated carbon and chitosan (Dai et al. 2013).

Effect of different nanoTiO 2 contents on AC/CS
It is well known that TiO 2 is the most efficient candidate for photocatalysis reactions and the addition at low concentration to CS/AC has led to an increase the photodegradation efficiency. The photocatalytic performances at different TiO 2  NP contents (0.2, 0.4, and 0.8%) were assessed by degrading RB dye under UV illumination. The adsorption of dye has been produced in dark and the data reveals that only 10-20% was adsorbed for all prepared TiO 2 percentages on CS/AC photocatalysts. Figure 7 (a, b) shows the photodegradation efficiency with different contents of TiO 2 NPs and the relation between RB concentration and illumination time within 120 min. The results showed that the different concentrations of TiO 2 displayed a higher value decolorization rate of the RB in the first stages of the degradation process and it will be decreased at a high concentration of dopant. This can be attributed to the fact that as the amount of dopant increases, the exposed surface-active area also increases. Noteworthy is that TiO 2 is a real catalyst in which it is a highly active, photostable, chemically stable, and highly porous structure. But after a certain limit, i.e. 0.4%, the further concentration of TiO 2 NPs is increased, and there was no increase in the exposed surface area of the photocatalyst (Gilja et al. 2019). This can be thought of as a saturation point; TiO 2 also increases the permeability of the supported materials thereby making it more effective for the decolorization of the Rose Bengal. Moreover, any more increase in the number of dopant contents had little or no effect on the degradation rate of the RB dye, since any increase in the TiO 2 NP percentage beyond this saturation point would only increase the thickness of the catalyst layer. This was confirmed by performing a reaction with a different ratio of denatured TiO 2 NPs from 0.2 to 0.8%. The limit of saturation point appeared at 0.4% of TiO 2 NPs after that increasing the amount led to aggregation and decreased the dye degradation (Sharma et al. 2013) as seen in Fig. 7 (a and b).

Evaluation of treatment method on the degradation of RB dye
Preliminary experiments were achieved to determine the photocatalytic performance Fig. 8 (a, b, and c). The effect of RB dye concentration keeps the catalyst loading concentration constant at 100 ml of dye solution, and the experiment is performed by adding 0.1 g/l of catalysts to different initial RB concentrations of 3.3, 5.5, and 7.9 ppm. This effect was implemented for higher TiO 2 NPs content (0.4%) as the best catalyst efficiency (AC / CS / TiO 2 (0.4%)). Figure 8a shows the degradation efficiency of the different primary dyes. The rate of photolysis of the RB dye depends on the potential for the formation of OH radicals on the surface of the catalyst and the interaction of the dye molecules with an OH-radical. The rate of photolysis decreased with increasing RB concentration. This is because as the number of RB dye molecules increases, the amount of light (the number of photons) that penetrates the dye solution to reach the surface of the catalyst decreases due to the obstacle in the light path. Thus, the optimum value of the catalyst and the dye concentration must be maintained, so that the maximum efficiency of hydrolysis can be achieved. (Gupta et al. 2012;Tang et al. 2015).
Three main removal treatments were tested during analysis for the efficacy of Rose Bengal degradation in wastewater. It should be noted that the first experiment was finished in the presence of a catalyst (AC/CS/TiO 2 (0.4%)) without UV light source based on the adsorption; the second process was performed in the presence of UV radiation without catalyst based on the photolysis; the third treatment was carried under UV radiation and in the presence of a catalyst based on photocatalysis. Rose Bengal under these processes for created composite catalyst was presented in Fig. (8b). It is worthy to mention that the effect of adsorption and photolysis is negligible (Aoudjit et al. 2018). The degradation percentage of selected dye under photocatalytic conditions in the presence of a tested catalyst for a reaction time of 120 min was 92.0%. However, during photolysis or adsorption, no allusion of effective elimination was observed, as indicated in Fig. 8b. Further conclusions of dye degradation and optimal conditions used for the operative removal of RB from contaminated water were taken. As seen in Fig. 8c, the photocatalytic activity of developed composite (AC/CS/TiO 2 (0.4%)) diminished capacity from 92 to 85.5% after five photodegradation cycles, which obviously and altogether illustrated the stability of the composite catalyst. The degradation efficiency of the developed photo-catalyst decreased during the 5th cycle, due to the aggregation of intermediates on active surface centers of the prepared composite. The obtained results confirmed the excellent reusability of prepared photoactive catalyst (AC/CS/TiO 2 (0.4%)) may well be

Kinetic model
The pseudo first-order kinetic model is shown in Fig. 9 by plotting Ln (a-x) against time (t). Where (a) is the initial concentration of the RB dye in (mg/l), x is the concentration at any other time t which is synonymous with the degradation rate, and k is a rate constant. K is the measure of the adsorption coefficient of the reactant onto the semiconductor particles and t is the reaction time. The adsorption coefficient of the reactant onto the semiconductor particles is measured by K, and the reaction time is measured by t. The experiments were carried out using 0.1 g of different catalysts with 7.9 ppm of RB dye concentration. It was found that the rate of reaction was increased when composite was used in comparison to CS/AC and TiO 2 catalysts, respectively, as indicated by the values of rate constant (k) (0.008, 0.01, and 0.02 min −1 ) for CS/AC, TiO 2 , and CS/ AC/TiO 2 (0.4%), respectively. The experiments have been carried out with 0.1 g of various catalysts and a concentration of 7.9 ppm of RB dye. As shown by the values of rate constant (k) (0.008, 0.01, and 0.02 min −1 ) for CS/AC, TiO 2 , and CS/AC/TiO 2 (0.4%), respectively, the rate of reaction was increased when composite was used in comparison to CS/AC and TiO 2 catalysts. In addition, Fig. 10 explains how Rose Bengal (RB) dye can degrade after being used in composites. After dispersing the CS/AC/TiO 2 in RB, the electrons on CS/AC should eventually transfer to the dye. Irradiated by a UV lamp, electrons (e-) in the valence band (VB) can be excited to the conduction band (CB) with the generation of the same amount of holes in the VB. Similarly, the photoinduced holes can be easily trapped by OH-to produce hydroxyl radical species further, which is a strong oxidant to partial or complete mineralization of dyes. The suggested mechanism of dye degradation can be described as following: Modifying the TiO 2 surface with CS/AC provides an effective environment for enhancing the dye interaction, which improves the photodegradation process. Additionally, chitosan and activated charcoal themselves adsorb dye molecules, which when combined with TiO 2 increases the efficiency of the composite.

Antibacterial activity
The antibacterial performance of a prepared CS/AC at different concentrations of TiO 2 NP composites was evaluated by the broth inhibition approach. From the data obtained in Table 5 and Fig. 11, we can observe that all the prepared composites with different concentrations of TiO 2 NPs showed antibacterial activity against reference strains used and the higher concentration of TiO 2 NPs with CS/ AC brought about the higher antibacterial activity for all model bacteria used. Based on the OD and percent of growth inhibition against the four models of bacteria, the higher antibacterial activities were observed for both B. subtilis and S. aureus followed by P. aeruginosa and then E. coli for all composites of CS/AC at different concentrations of TiO 2 NPs. We can conclude that the prepared composites of CS/AC at different concentrations of TiO 2 NPs have high antibacterial activity and inhibit the growth of bacteria and are therefore believed to have great potential for use as antibacterial composites. Due to its chemical stability, non-toxicity, and availability, TiO 2 has been demonstrated as a favorable antibacterial agent and fabricated with CS in several studies According to their chemical stability, nontoxicity, and availability of TiO 2 , it was demonstrated as a favorable antibacterial agent and fabricated with CS in several studies (Raut et al. 2016;Li et al. 2019b;Moqeet Hai et al. 2019). Other studies reported the action of AC with TiO 2 as antimicrobial agents (Yang et al. 2012;Ren et al. 2020).

Larvicidal and pupicidal activity
The CS/AC/TiO 2 NP synthesized was effective with the larvae and pupae of C. pipiens. The larvae of C. pipiens were present as highly susceptible to the synthesized CS/ AC/TiO 2 than the pupae at the same concentrations of TiO 2 NPs. The mortality could be observed after 24 h of treatment. The larvae of C. pipiens were found highly susceptible to the synthesized CS/AC/TiO 2 (0.4%) than the same larvae at CS/AC/TiO 2 (0.2 and 0.8%). The mortality was recorded after 24 h. The soon three instars of C. pipiens were observed for more susceptible to the synthesized CS/ AC/TiO 2 and exhibit high mortality after 24 h of treatment while the fourth larval instar and pupae were low susceptible to the synthesized CS/AC/TiO 2 . The high value for larval  pipiens when compared to their corresponding nanoparticles counterpart. The mechanism which causes the death of the larval instars and pupae could be due to the capability of the NPs to permeate out of the larval membrane. The silver nanoparticles in the intracellular space can link to sulfur-containing proteins or phosphorus including component as DNA, leading to the denaturation of some organelles and enzymes. Thereafter the decrease in membrane permeability and disturbance in proton motive force causes loss of cellular function and finally cell death. Silver nanoparticles in the intracellular space can bind to sulfur-containing proteins or phosphorus-containing components such as DNA, causing organelles and enzymes to denaturate. The loss of cellular function and, eventually, cell death is caused by a decrease in membrane permeability and a disruption in the proton motive power (Jawad and Nawi 2012). At the different doses of CS/AC/TiO 2 (0.2, 0.4, and 0.8%), all tested starved females of mosquito vector C. pipiens exhibited repellency activity ( Table 6). The repellent activity was varied according to their concentration. Overall, based on the record, all doses of CS/AC/TiO 2 (0.4%) were more effective in exhibiting the repellent action against C. pipiens starved females than other materials. Similar results were also recorded by Deepalakshmi and Jeyabalan (2017) who tested the repellent activity of Glochidion neilgherrense, Cinnamomum wightii, and Leucas linifolia methanol leaf extracts against C. quinquefasciatus and they found that all tested concentration promising mosquito repellency properties.

Conclusion
In this work, the fabrication of crosslinking CS / AC and CS / AC / TiO 2 nanocomposite as sustainable materials for the removal of anionic dyes, such as Rose Bengal (RB) dye, assess its photodegradation properties. The synthesized nanocomposites were characterized with different techniques such as SEM, FT-IR, and XRD. The photo-degradation of RB shown the high efficacy of nanocomposites CS/ AC/TiO 2 NPs in dye as removal compared to CS/AC. The antibacterial activities of the fabricated CS/ AC/ at different concentrations of TiO 2 NPs exhibit antibacterial activity against all the model bacteria used. CS/AC/0.4% TiO 2 NPs showed maximum efficacy against larvicidal activity and pupicidal of mosquito vector which recorded 99.00 ± 1.14, 95.00 ± 1.43, 92.20 ± 2.64 for first, second, and third larval